Standard in vitro fertilization procedures cannot preserve the reproductive potential of women in all situations, such as premature ovarian failure or oncotherapy induced sterility. The in vitro maturation of granulosa cell-oocyte complexes (GOC) may provide an alternative to current methodology. The goal of this project is to employ a three-dimensional, engineered, synthetic stroma to examine GOC maturation and development in vitro. The central hypothesis underlying this proposal is that the development of the granulosa cells and the oocyte must be coordinated to allow effective maturation of the oocyte to provide fertilization. Oocyte maturation and granulosa cell development involve endocrine, paracrine, and autocrine-acting factors in addition to appropriate somatic-germ cell and cell-matrix interactions. The native stroma surrounding a GOC dynamically regulates growth and maturation by maintaining cellular interactions and providing the matrix interactions that direct cell function. We will employ an atginatebased scaffold as a synthetic stroma that provides the factors that stimulate development and removes the factors that inhibit maturation. Alginate exhibits minimal cellular interactions and thus provides an environment that can be designed to present specific stimuli. Preliminary results have demonstrated gentle encapsulation of individual GOCs within beads and that cultured complexes retain their normal architecture. GOCs produce estradiol in response to growth and differentiation factors. Importantly, retrieved oocyte undergo germinal vesicle breakdown and fertilization results in the development to the two-cell stage. We specifically aim to use this system to examine the coordination of GOC development and oocyte maturation by 1) mechanically supporting the GOC to maintain the cell-cell interactions 2) supplying growth and differentiation factors, and 3) regulating the granulosa cell-matrix interactions. We systematically examine the role of each component in directing the cellular processes within the developing GOC, and thereby the ultimate maturation of the oocyte in vitro. This three-dimensional culture system may provide novel therapeutic approaches for germline preservation.